Molecular biological studies on etiologies of hereditary neurological diseases.
| Project/Area Number |
62440039
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| Research Category |
Grant-in-Aid for General Scientific Research (A)
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| Allocation Type | Single-year Grants |
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| Research Field |
Neurology
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| Research Institution | Niigata University |
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Principal Investigator |
MIYATAKE Tadashi Brain Research Institute, Niigata University, Professor, 脳研究所, 教授 (50048998)
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| Co-Investigator(Kenkyū-buntansha) |
SATO Shuzo University Hospital, Niigata University, Lecturer, 医学部附属病院, 講師 (30115034)
ATSUMI Tetsushi Brain Research Institute, Niigata University, Assistant Professor, 脳研究所, 助教授 (50049061)
TSUJI Shouji University Hospital, Niigata University, Assistant, 医学部附属病院, 助手 (70150612)
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| Project Period (FY) |
1987 – 1988
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| Project Status |
Completed (Fiscal Year 1988)
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| Budget Amount *help |
¥24,000,000 (Direct Cost: ¥24,000,000)
Fiscal Year 1988: ¥5,000,000 (Direct Cost: ¥5,000,000)
Fiscal Year 1987: ¥19,000,000 (Direct Cost: ¥19,000,000)
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| Keywords | Sialidosis / Sialidase / Sphingolipidosis / Molecular cloning / cDNA / cDNAクローニング / ライソソーム |
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| Research Abstract |
A sialidase deficiency has been found in various deseases including mucolipidosis I, cherryred spot myoclonus syndrome and galactosialidosis. The molecular mechanisms of the sialidase deficiency, however, have not been elucidated, primarily due to the extreme difficulty of the purification of the human sialidase. In order to investigate the molecular mechanisms of the sialidase deficiency, we have purified a sialidase from human placenta. The purified active sialidase consists of 5 proteins with Mr. of 78kDa, 64kDa, 46kDa, 30kDa and 20kDa. of these, 64kDa, 30kDa and 20kDa proteins have been assigned to -galactosidase and protective protein. Our strategy for molecular study of human sialidase has been to isolate cDNA clones for 78kDa and 46kDa proteins, which are the best candidates for the sialidase.
As the first step for the molecular cloning of the sialidase proteins, we purified the 46kDa protein to homogeneity and have determined the partial amino acid sequence of tryptic peptides of the 46kDa protein. Utilizing the amino acid sequences, we synthesized two oligonucleotide probes and screened a cDNA library. One clone (]sy.lbdabar.[)HS1013) hybridized to both oligonucleotides. Nucleotide sequence analysis of the cDNA showed complete-co-linearity of the nucleotide sequence with chemically determined amino acid sequence. Using the cDNA as a probe, we have succeeded in isolating a fulllength cDNA for the 46kDa protein. With the full-length cDNA for the 46kDa protein, we are currently looking at the function of the 46kDa protein by introducing the cDNA into mammalian cells. We are also doing Southern and Northern analyses to see if there are any mutations in the 46kDa protein gene. These approach should give us better understanding of the molecular mechanisms of sialidase deficien cies in human.
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Report
(3 results)
Research Products
(9 results)
